Download Extracting DNA from cheek cells

Extracting DNA from cheek cells:
a classroom experiment for Year 7
upwards
Dr Kathryn Scott
Research Administrator, Zitzmann Group, Department of
Biochemistry
Lecturer in Biochemistry, Christ Church
Extracting Human DNA in the Classroom
• Buccal (cheek cells) can be harvested painlessly and
in sufficient quantity to visualise DNA extracted in a
simple 4-step protocol
• We will be carrying out an optimised DNA
extraction and discussing ‘kitchen chemistry’
alternatives to the materials used
• DNA extraction based on:
R.P. Hearn & K.E. Arblaster. DNA Extraction Techniques for
Use in Education (2010) Biochem Mol Biol Edu 38(3) 161166
• Original optimised protocol requires a centrifugation
step
The Steps in DNA Extraction
1. Cell Harvesting
3. Protein Digestion
2. Cell Lysis
4. DNA Precipitation
Objectives
• Basic level students will
• Know that DNA is found in the nucleus of cells
• Learn how to extract DNA from cells and describe the
purpose of the key steps of cell lysis, protein
degradation and DNA precipitation
• Observe the appearance of human DNA
• More advanced students will also
• Learn why buccal cells are a good choice for this
experiment
• Understand the role of SDS and EDTA in cell lysis
• Understand the role of salt and alcohol in DNA
precipitation
Risk Assessment
• Biological samples should only be handled by the person
from whom they are taken
• Lysis buffer is an emetic and may cause irritation if in
contact with skin or eyes
• Protease solution may cause irritation if in contact with skin
or eyes
• Isopropyl alcohol is toxic if consumed and if absorbed
through the skin
Step 1 – Cell Harvesting
Step 1 – Harvesting Cells
• Pipette 3 ml water into a drinking cup
• Gently chew the inside of your mouth for 30 seconds
• blood doesn’t help!
• Take the water from the cup into your mouth and move it
around for 30 seconds
• Don’t swallow the water
• Carefully spit the water into a test tube
Step 2 – Cell Lysis
Step 2 – Cell Lysis
• Add 2 ml of lysis buffer to the test tube you will be using for
DNA extraction
• Pour the contents of your cup into the test tube
• Put the cap on your tube
• Gently swirl the tube to mix
• Shaking shears the DNA leading to short strands at the
end of the experiment
Step 3 – Protein Digestion
Step 3 – Protein Digestion
• Add 0.25 ml (~5 drops) of Proteinase K solution to the tube
• Adding an excess does not cause any problems
• Put the cap on your tube
• Gently swirl the tube to mix
• Place your tube in the 56oC water bath for 10 minutes
Buccal Cells Provide An Excellent Source of DNA
Stratified squamous
non-keratinized
epithelium
Connective tissue
50 mm
Buccal Cells Provide An Excellent Source of DNA
50 mm
Cell Lysis Buffer
• 50 mM Tris pH 8.0
• Buffering for DNA stability and optimal enzyme activity
• 1 % Sodium dodecyl sulfate (SDS) (sodium lauryl
sulfate)
• 1 mM Ethylenediaminetetraacetic acid
Cell Lysis – The Structure of SDS Micelles
hydrophilic
hydrophobic
Sodium
Dodecyl
Sulfate
(SDS)
Computer
simulation of a
Sodium Dodecyl
Sulfate
Micelle
Micelle
Cross-section
The Structure of Cell Membranes
hydrophilic
hydrophobic
The lipid 1-palmityl-2-oleoylphosphatidylcholine
(POPC)
Cross section from a
computer simulation of a
pure POPC bilayer
SDS Disrupts Cell Membranes
+
SDS
micelle
Lipid
bilayer
Mixed
micelle
A concentration of 0.3% - 1% SDS is sufficient to disrupt the membranes
of buccal cells
Cell Lysis – What Does EDTA Do?
Ethylenediaminetetraacetic Acid
EDTA Inhibits Enzymes such as DNase I
DNA
Ca2+
Mg2+
DNase I
from
bovine
pancreas
Both Ca2+ and Mg2+ are essential
for DNase I function
DNase enzymes are found in most cells
Discussion Point
• Given that the lysis buffer is very similar in
composition to shampoo, why does shampoo not
lyse our skin cells
Stratified squamous
keratinized
epithelium
The skin has a protective layer
known as the Stratum Corneum.
The Stratum Corneum consists
of cells that have have lost their
nuclei, are embedded in a lipid
matrix and are enriched in
keratin proteins.
"Epidermal layers" by Mikael Häggström, based on work by Wbensmith File:WVSOM Meissner's corpuslce.JPG at Wikimedia commons
Discussion Point
Keratinized epithelial (skin
cells) stained to visualise the
DNA (green) and keratin
filaments (red)
Note – these cells are from
the lower epithelial layers
Keratin has several important
roles
• Strengthens Cells
• Acts like a molecular
sponge absorbing water if
skin is immersed in water
for a long time
https://commons.wikimedia.org/wiki/File:Epithelialcells.jpg
Proteinase K Digestion
• Many proteins precipitate under the same
conditions as DNA
• If we digest the proteins into amino acids then only DNA
will precipitate
Protein digestion also removes the histone ‘cotton reels’ around which the DNA is wrapped
Proteinase K Digestion
• Originally extracted from the fungus Tritirachium album
• Named due to its ability to cleave Keratin
• Many proteinases only cleave after a specific amino
acid
• This leads to the production of large fragments
• Proteinase K is relatively non-specific, therefore leaving very
small fragments
• Is active over a wide range of temperatures
• Is active in the presence of a wide range of additives
including
• SDS
• EDTA
Step 4 – DNA Precipitation
Step 4 – DNA Precipitation
• Swirl your tube gently to mix
• Hold your tube at 45o and carefully pour in 10 ml of cold
isopropyl alcohol
• Leave the tube on the desk for 5 minutes
• It is very important not to shake the tube
• After 5 minutes DNA should have precipitated at the
interface between the lysis buffer and the alcohol
• Swirling so that a vortex forms can aid precipitation
• Do not shake or invert the tube
DNA Precipitation
• DNA is a highly polar molecule
There is a negatively charged
phosphate group joining
every base in a DNA chain.
https://commons.wikimedia.org/wiki/File:DNA_chemical_structure.svg
DNA Precipitation
• When DNA molecules and NaCl are dissolved in
water the DNA, Na+ and Cl- ions will all be
surrounded by water molecules
• Water screens the charges on the DNA and salt ions and
prevents them interacting to form strong ionic bonds
• Adding ethanol disrupts the structure of water
around the ions, reducing the screening
• The positively charged Na+ ions and negatively charged
DNA phosphate groups interact to form strong ionic
bonds
• Many ions coming together leads to precipitation
Variations on the Protocol
• The optimised protocol has proven effective in a
classroom setting with students as young as Year 5
• Cost per student is still high – but talk to your local
University
• SDS - £27.50 per 25 g – need 1 g per 100 ml buffer (2ml required
per student) Sodium lauryl sulfate is cheaper
• EDTA - £14.50 per 100 g – need 29 mg per 100 ml buffer
• TrisHCl - £37.50 per 100 g – need 0.8 g per 100 ml buffer
• 100 ml Tris-EDTA buffer pH 8 (10 mM Tris, 1 mM EDTA) - £19.50
(works well)
• 100 ml 100x Tris-EDTA buffer pH 8 (1 mM Tris, 0.1 mM EDTA) £18.10
• ProteinaseK – 10 mg - £23.00
Variations on the Protocol
• Cell harvesting – scraping vs chewing
• Lysis buffer – Tris-EDTA-SDS vs showergel and hand
soap
• Enzyme – Proteinase K vs no Enzyme vs contact
lens tablets (Subtilisin A)
• Ethanol vs Isopropanol
Variations - Cell Harvesting
• Harvesting sufficient buccal cells is essential for
successful DNA extraction
Chewing Cheeks
• Isotonic vs non-isotonic solutions
Scraping Cheeks
Variations – Lysis Buffer
Variations – Lysis Buffer
Tris pH 8.0, 1% SDS,
1 mM EDTA
NO SHAKING
5% Handwash
NO SHAKING
5% Shower Gel
NO SHAKING
Variations Proteinase
• Proteinase K is active under a wide range of
conditions but is only available from specialist
manufacturers
• Other proteinases are more readily available
• Subtilisin A – contact lens cleaner
• Less expensive than proteinase K ~£10 for a class of 30
• not compatible with EDTA, reduced activity in SDS, optimal
temperature not stated on packaging
• Meat tenderiser
• May contain one of a variety of enzymes
• May be contaminated with DNase (proved to be the case in our
experience)
Variations – Protease
Poor DNA
yield
Proteinase K
NO SHAKING
Subtilisin A
No EDTA
37oC
NO SHAKING
No Protease
Sample 1
NO SHAKING
No Protease
Sample 2
NO SHAKING
Variations – Isopropanol vs Ethanol
• DNA is less soluble in isopropanol than ethanol
• therefore a lower volume of isopropanol is required for
DNA precipitation
• Isopropanol is much more toxic than ethanol
• drinking 10 ml of isopropanol could prove fatal
• Isopropanol is also readily absorbed through the skin
• The benefit of an increase in yield when using
isopropanol must be carefully evaluated against the
increased risk
Variations – Isopropanol vs Ethanol
Isopropanol
NO SHAKING
Ethanol
NO SHAKING
Pitfalls – Harvesting Sufficient Cells is Vital
DNA from a
thorough cell
harvest.
Tris pH 8.0, 1% SDS, 1 mM EDTA
Proteinase K
Isopropanol
NO SHAKING
DNA from a
second round
of cell
harvesting
immediately
after the first.
Tris pH 8.0, 1% SDS, 1 mM EDTA
Proteinase K
Isopropanol
NO SHAKING
Pitfalls – Large sample volume
Proteinase K
AFTER SWIRLING
No Protease
Sample 1
AFTER SWIRLING
No Protease
Sample 2
AFTER SWIRLING
Conclusions
• Human DNA extraction can be carried out in a 45
minute lesson for lower years
• Upper years benefit from an additional theory lesson
• Upper years can relate the practical to a range of
different areas of the curriculum
•
•
•
•
Tissue formation
DNA structure and function
Enzymes
Solubility